Studies of E2F and RB-family proteins highlight the importance of these proteins in the regulation of mammalian cell proliferation. While the significance of these proteins is well established, our understanding of what these proteins do, and why their activities are so important, remains vague. At least 119 different cellular proteins have been reported to interact with pRB. E2F is the best known partner of pRB, but it remains uncertain whether E2F is the main target of RB action, one of several targets, or one of a large number of pRB-associated factors that have important roles in different settings. Compounding this problem, the number of proteins linked to pRB in the literature steadily continues to rise. In a similar way, the rapidly growing E2F literature emphasizes that E2F is a complex mix of activities that are subject to many tiers of regulation. Recent studies have shown that E2F is able to induce the expression of a large number of genes that affect a broad cross-section of cellular functions. In order to complement studies of RB and E2F proteins in mammalian cells we have identified and characterized their Drosophila homologs. Our initial experiments show that flies contain both activator and repressor E2Fs, and that the overexpression of dE2F1 is able to drive cell proliferation and induce apoptosis in ways that closely resemble mammalian E2F proteins. One of the major advantages of Drosophila is the opportunity to perform genetic screens. Given that the role of RB and E2F homologs in flies so closely resembles their mammalian counterparts, these screens provide an opportunity to identify, in a relatively unbiased way, those activities that have a significant impact on RB/E2F function in vivo. In this application we propose to screen for mutations that specifically suppress Drosophila phenotypes that have been generated by raising the levels of E2F and RBF. Mutations isolated in this way will be sorted into those that modify E2F-induced phenotypes in multiple settings, and those that are tissue or stage specific. We will test whether the modifiers have a direct effect on transcription, and we will identify modifiers that selectively affect E2F-induced apoptosis. Human homologs will be sought and tested for the ability to act on E2F or pRB in mammalian cells. Since deregulated E2F activity is thought to drive the inappropriate proliferation of many tumor cells, genes that have a strong and conserved impact on pRB and E2F activity will be assessed for mutations in tumor cells.